High Risk HPV Oncogene E7 Upregulates miR-182 through TGF-¦Â/Smad Pathway

• Other Authors: Chen, Jiao (author.)
• Format: Others
• Language: English; Chinese
• Published: 2016
• Online Access: http://repository.lib.cuhk.edu.hk/en/item/cuhk-1292220

高危型人乳頭狀瘤病毒（HPV）是引起宮頸癌最重要的因素。HPV基因，比如E6和E7癌基因，與細胞增殖轉化密切相關。E6基因產物能夠與抑癌基因p53結合並誘導其降解阻斷凋亡；E7基因產物能夠與抑癌基因pRb結合促使細胞持續進入S期而永生化。然而，低危型HPV,如HPV6b&11,常見於尖銳濕疣，卻很少引起惡性病變。就目前所知，低危型HPVE6蛋白並不能結合p53，E7蛋白與pRb的親和力也較低。而有證據表明僅僅高危型HPV感染也不足以引起惡性病變，其它的宿主基因的變化對引起宮頸癌也是非常重要的。 === 微型核糖核酸(microRNAs)在細胞調節過程中是非常重要的，並且被發現在多種癌症中表達異常。在我們過去的研究中，鑒定了一系列異常表達的miRNAs，並發現miR-182在宮頸癌細胞系及宮頸癌組織樣本中高表達。功能研究表明miR-182 能夠通過干擾細胞增殖誘導凋亡的機制調節作用于宮頸癌。是否HPV與miR-182有關卻仍然不清楚。 === 為了確定HPV與miR-182之間的關係，我們設計了一系列實驗。在含有不同亞型的高危型HPV(16,18,31,52and58)E7的細胞系中，通過qRT-PCR/Weston-blot檢測發現，過表達/沉默高危型HPVE7基因可引起TGF-β和miR-182的上調/下調，並且TGF-β能夠引起miR-182的表達變化(P<0.05)，并通過免疫組織化學/原位雜交技術，發現TGF-β和miR-182在高危型HPV宮頸癌組織樣本中高表達；利用染色質免疫共沉澱和荧光素酶分析技術預測並驗證了E2F1蛋白能夠通過與TGF-β啟動區結合誘導TGF-β的表達；同時Smad4蛋白與miR-182啟動區結合誘導miR-182的表達。另外，我們在裸鼠腫瘤模型中也驗證了HPV E7 - TGF-β - miR-182調節通路的存在。通過共培養系統，我們還觀察到HeLa細胞能夠誘導附近正常細胞的變化。 === 這些發現給假設奠定了一個堅實的基礎：宮頸癌中，高危型HPVE7能夠通過TGF-β通路調節miR-182，這可能是一條由高危型HPV癌基因誘導宮頸癌的重要通路。本研究可能為進一步探究高危型HPV引起宮頸癌的分子機制提供新的視角。 === 關鍵字：人乳頭狀瘤病毒，微型核糖核酸，宮頸癌，miR-182，TGF-β === The high risk human papillomavirus (HPV) is the single most important etiological agent in cervical cancer. The HPV genes, such as E6 and E7 oncogenes, are closely related to cellular transformation and immortalization. The E6 gene product binds to the p53 tumor suppressor gene and induces its degradation to block apoptosis; the E7 gene product targets and binds to the tumor suppressor gene pRb and consequent entry into S phase to immortalize. However, low risk HPV, such as HPV 6b & 11, which are the commonest HPV subtypes causing condyloma acuminatum (CA), rarely induce malignant changes. It is known that low-risk HPV E6 protein does not bind p53, and E7 gene product has a small affinity with pRb. However, evidence suggests that high risk HPV infection alone is insufficient to induce malignant changes, and other host genetic variations are important in the development of cervical cancer. === MicroRNAs (miRNAs) are very important in the cellular processes and found to be dysregulated in many cancers. In our previous study, we identified several aberrantly expressed miRNAs and found that miR-182 is significantly up-regulated in cervical cancer cell lines and cervical carcinoma. Functional analysis showed that miR-182 could work as an onco-miRNA by disrupting cell proliferation and inducing apoptosis. Whether HPV is associated with miR-182 is still unclarified. === In order to determine the relationship between HPV oncogenes and miR-182, we performed a series of experiments. We found that overexpression/knockdown regulation of HPV E7 gene caused an increase/decrease of TGF-β and miR-182 expression, and TGF-β could induce the expression of miR-182 in cells with different high risk HPV E7 (16, 18, 31, 52 and 58) by qRT-PCR and Weston-blot (P<0.05), and TGF-β and miR-182 were overexpressed in primary cervical carcinoma with high risk HPV by immunohistochemistry and in situ hybridization. We also validated the binding activity of E2F1 and Smad4 to the promoter region of TGF-β and miR-182, respectively, which could induce the expression of TGF-β and miR-182 by ChIP-PCR and luciferase assay; and the HPV E7 - TGF-β - miR-182 pathway was also validated in tumor xenograft nude mice. By co-culture system, we also observed HeLa cells could induce some changes in the surrounding stromal cells. === These findings provided a solid foundation that miR-182 is regulated by high risk HPV E7 via TGF-β pathway in cervical cancer which may be an important pathway for the pathogenesis of cervical cancer induced by high risk HPV oncogene. This will provide new insight to explore molecular pathways of high risk HPV-induced cervical cancer in understanding the development of cervical cancer. === Keywords: Human Papillomavirus, MicroRNA, Cervical Cancer, miR-182, TGF-β === Chen, Jiao. === Thesis Ph.D. Chinese University of Hong Kong 2016. === Includes bibliographical references (leaves ). === Abstracts also in Chinese. === Title from PDF title page (viewed on …). === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only.